Effects and Eradication of Mycoplasma Contamination on Patient-derived Colorectal Cancer Organoid Cultures

Patient-derived organoids are a useful platform for identification and testing of novel precision oncology approaches. Patient-derived organoids are generated by direct culture of patient samples. However, prior to development into patient-derived organoids, these samples are often processed for clinical use, opening the potential for contamination by Mycoplasma and other microbes. While most microbes can be detected by visual inspection, Mycoplasma can go undetected and have substantial impacts on assay results. Given the increased use of patient-derived organoids, there is a growing need for a standardized protocol to detect and remove Mycoplasma from organoid models. In the current study, we report a procedure for Mycoplasma removal by passaging organoids through mice as patient-derived organoid xenografts. In vivo passage of patient-derived organoids followed by re-establishment was 100% effective at decontaminating colorectal patient-derived organoids (n = 9), based on testing with the Sigma LookOut Mycoplasma PCR Detection Kit. This process can serve as a method to re-establish contaminated patient-derived organoids, which represent precious models to study patient-specific genomic features and treatment responses. Significance: Organoids are valuable models of cancer. Mycoplasma contamination can alter organoid drug sensitivity, so there is a need for a standardized protocol to detect and remove Mycoplasma from organoids. We report a simple procedure for removing Mycoplasma from organoids via in vivo passaging through mice followed by re-establishment of organoids.


Introduction
Patient-derived models of cancer, such as cell lines, organoids, and patientderived xenografts (PDX) are increasingly being utilized as preclinical models to facilitate the identification and development of new therapeutics.Patientderived organoids (PDO) can accurately model the biology of patient tumors, both at the phenotypic and genotypic levels (1)(2).Furthermore, PDO have been used to perform high-throughput drug screens and correlate with patient therapeutic response, making them a useful platform for precision oncology approaches to identify novel therapies (1)(2).
While organoid models have clear potential to aid in preclinical development of new therapies, these patient-derived models are also susceptible to contam-ination during clinical processing.One particularly difficult contaminant is Mycoplasma.Mycoplasma are the smallest bacteria that can self-replicate and be less than 1 μm in size (3)(4).Because of their small size and antibiotic resistance (3), they are frequently found as contaminants in cell culture (3)(4).Mycoplasma can negatively affect eukaryotic cells in numerous ways, from altering DNA, RNA, and protein levels to changes in cell growth and viability (3,(5)(6).Proper aseptic techniques can prevent Mycoplasma contamination, but if there are no backup stocks for an infected line, eradicating Mycoplasma may be difficult.The most commonly-used method for eliminating Mycoplasma from cultures includes various antibiotics, such as quinolones and BM-Cyclin, as well as Plasmocin and Plasmocure (3,(7)(8)(9).While antibiotics may kill Mycoplasma, they can also be extremely stressful to cancer cells due to their mechanisms, such as inhibiting protein synthesis or inducing double-strand DNA breaks (3).Another concern is that Mycoplasma may develop resistance to antibiotic treatments, thereby making it more difficult to eliminate.Detection and treatment of Mycoplasma have been extensively studied in a variety of cancer cell lines (3,(7)(8)(9)(10)(11).As researchers turn to other patient-derived models of cancer more representative of tumors in the human body, proper decontamination methods need to be established for these models as well.To date, no studies have extensively shown the effects of Mycoplasma contamination on PDOs or how they can be successfully treated.Thus, there is a growing need for a standardized method of removing Mycoplasma from organoid cultures without compromising cells of interest.Here, we demonstrate the effects of Mycoplasma in colorectal cancer PDO lines and establish a standard protocol for decontaminating Mycoplasma from organoid lines based on testing with the Sigma LookOut Mycoplasma PCR Detection Kit.

Establishment and Maintenance of PDOs
The study was conducted in accordance with the U.S. Common Rule.Colorectal cancer patient tissue samples were collected with written informed consent under a Duke Institutional Review Board-approved protocol (Pro00089222), obtained from either the NCI's Cooperative Human Tissue Network or through Duke University BioRepository & Precision Pathology Center and written consent was obtained from each subject.Tissue samples were cut into pieces approximately 2 mm 3 with a sterile scalpel and mechanically digested in C-tubes with 10 mL of DMEM using a gentleMACS Dissociator (Miltyenyi Biotec).The three protocols on the gentleMACS Dissociator for digesting human tumors, h_tumor_01, h_tumor_02, and h_tumor_03, were each performed twice.Cells and tissue fragments were filtered through 70 μm filters and centrifuged at 500 × g for 5 minutes.Supernatants were aspirated and a total of 1.25 × 10 5 cells were plated in 50 μL domes composed of 30% cell suspension in media and 70% Matrigel (Corning).

Treatment of PDOs with Antibiotics
Media was removed from organoids and replaced with fresh media containing Plasmocin (Invivogen) according to manufacturer's protocols.Following treatment, organoids were tested for Mycoplasma as stated above.

Elimination of Mycoplasma by Passaging Through Mice
A total of 2 × 10 6 cells from each Mycoplasma-positive colorectal cancer organoid were subcutaneously injected into JAX NOD.CB17-PrkdcSCID-J mice.After the tumors grew to approximately 0.5 cm 3 , mice were euthanized following Duke Institutional Animal Care and Use Committee (IACUC)approved protocols, and the tumor was harvested.Tumors were mechanically digested in C-tubes with 10 mL of DMEM using a gentleMACS Dissociator (Miltyenyi Biotec) and the m_impTumor_01.01 protocol was performed twice.
Cells and tissue fragments were filtered through 70 μm filters and centrifuged at 500 × g for 5 minutes.The supernatants were aspirated, and a total of 1.25 × 10 5 cells were plated in 50 μL domes composed of 30% cell suspension in media and 70% Matrigel (Corning).In vivo passaged colorectal cancer PDO were maintained in colorectal cancer media as described above.Once organoids grew, they were authenticated to be human cells.

Drug Treatment Dose-response Curves
Stock solutions for oxaliplatin, SN38, and 5-fluorouracil (5-FU) were prepared at 10 mmol/L in PBS, DMSO, and PBS, respectively.Once the organoids were confluent, media was aspirated, and 1 mL of PBS was added to each well to detach Matrigel domes.Matrigel was centrifuged at 750 × g for 5 minutes.A total of 1 mL of TrypLE Express (Gibco) was used to dissolve Matrigel and break down organoids.These mixtures were incubated for 5 minutes, and TrypLE was neutralized by adding 5 mL of DMEM F12 media with 10% FBS and 1% penicillin/streptomycin.After centrifuging at 750 × g for 5 minutes, supernatants were aspirated.Cells were plated in 96-well plates in 5 μL domes at a concentration of 2 × 10 3 cells per well.Organoids were allowed to recover for 2 days before addition of drugs.
To add drugs, media was aspirated from all wells and replaced with colorectal cancer media containing 2X of each component in the RealTime Glo MT Cell Viability Assay kit (RTG; Promega).Organoids were treated with each of the three compounds in a nine-point dilution series with a dilution factor of three starting from 1 mmol/L for oxaliplatin, 8 μmol/L for SN38, and 1 mmol/L for 5-FU, with five replicates per dose.Fluorescence was measured every day for 3 days using a Varioskan Lux plate reader (Thermo Fisher Scientific).Plates were imaged using an Incucyte S3 live cell imaging system.IC 50 values were calculated using a nonlinear curve fit with the log (inhibitor) versus response (three parameters) function in GraphPad Prism (GraphPad Prism, RRID:SCR_002798).

High-throughput Drug Screens and Growth Studies
The NCI Oncology panel of 147 FDA-approved drugs was provided in 96-well plates by the Duke Functional Genomics Core Facility and tested in triplicate.Once the PDO were confluent, media was aspirated, and 1 mL of PBS was added to each well to detach the Matrigel domes.Matrigel was centrifuged at 750 × g for 5 minutes.A total of 1 mL of TrypLE Express (Gibco) was used to dissolve Matrigel and dissociate organoids.These mixtures were incubated for 5 minutes and TrypLE was neutralized by adding 5 mL of DMEM F12 media with 10% FBS and 1% penicillin/streptomycin.After centrifuging at 750 × g for 5 minutes, supernatants were aspirated.PDO cell suspensions were used to make MicroOrganoSpheres (MOS) as described previously (15).A total of 100 MOS were plated per well.Cell viability was assessed using the Cell Titer-Glo luminescent Cell Viability Assay kit (Promega) after 72 hours.Plates were imaged using an Incucyte S3 live cell imaging system.Percent killing was calculated as follows: 100*[1 − (average CellTiterGlo drug /average CellTiterGlo DMSO )].
PDO cell suspension was also plated in triplicate 5 μL domes in 96-well plates at a concentration of 2 × 10 3 cells per well.Plates were imaged every other day for 14 days using an Incucyte S3 live cell imaging system or ImageXpress

Data Availability
The data generated in this study are available upon request from the corresponding author.

Effects of Plasmocin on Mycoplasma Clearance and Growth Rates in Colorectal Cancer PDO Cultures
Several methods have been established to limit or remove Mycoplasma from cell cultures, including antibiotics and coculturing with macrophages.Currently, antibiotics remain the most common method for eliminating Mycoplasma from cultures (3,(7)(8).To test the effectiveness of antibiotics on Mycoplasmapositive organoid cultures, we treated four patient-derived colorectal cancer PDO lines with Plasmocin in triplicate for 2 weeks according to the manufacturer's protocol.Treatment of organoids with Plasmocin had mixed results.In PDO1, two of three replicates converted to Mycoplasma-negative while PDO2 and PDO3 remained Mycoplasma positive (Fig. 1A), based on testing with the Sigma LookOut Mycoplasma PCR Detection Kit.Sample PDO4 was the only line that became Mycoplasma negative giving an overall conversion rate of 25% (1/4; Fig. 1A).
Because PDO4 was the only sample to be fully cleared by Plasmocin treatment, we evaluated the growth rate of PDO4 after treatment with Plasmocin by quantifying Cell Titer Glo fluorescence of the organoids over 2 weeks (Fig. 1B).The organoids produced the highest signal at the beginning and had a lower signal with every subsequent measurement, suggesting that Plasmocin negatively impacted the growth of PDO4 organoids (Fig. 1B).

Converting Mycoplasma-positive PDOs to Mycoplasma-negative PDO by Passaging Through Mice
We next tested whether passaging organoids through mice may remove Mycoplasma without the need for antibiotic treatments.To do this, we passaged colorectal cancer PDO lines through immunodeficient mice (JAX NOC.CB17-PrkdcSCID-J mice) as patient-derived organoid xenografts.We tested this system on n = 9 Mycoplasma-positive colorectal cancer organoids by inoculated these cells subcutaneously into the flanks of JAX NOC.CB17-PrkdcSCID-J mice.Tumors were followed until they reach a size of approximately 0.5 cm 3 .Tumors were then harvested and regrown as organoids.All lines were authenticated after growing as organoids (Supplementary Fig. S1).Using this method, we achieved a 100% Mycoplasma clearance rate from the organoid lines based on testing with the Sigma LookOut Mycoplasma PCR Detection Kit (Fig. 2A).This kit tests for all seven Mycoplasma species that account for 95% of Mycoplasma contamination, in addition to 12 other species (12)(13)(14).On the basis of these results, we have formulated an easily-adoptable protocol for decontaminating Mycoplasma-positive colorectal cancer PDO as outlined in Table 1.

Mycoplasma Contamination can Alter Organoid Growth and Drug Sensitivity
Quantification of growth rates of Mycoplasma-positive and -negative organoids by Cell TiterGlo indicated significant differences in matched Mycoplasmanegative and -positive lines, with CTG values at least five times higher in the Mycoplasma-negative lines (Fig. 2B).This suggests that Mycoplasma negatively impacts growth and viability of organoids and underscores the need to confirm Mycoplasma status in organoid models.Unlike eradication with antibiotics, passaging organoids through mice does not hinder the growth of the PDO.We next wished to determine whether Mycoplasma may impact organoid drug sensitivity.To do this, we performed dose-response curves for three specific standard-of-care drugs (oxaliplatin, irinotecan, and 5-FU) used in the treatment of colorectal cancer and found a significant difference in drug sensitivity between the Mycoplasma-positive and -negative colorectal cancer organoids (Fig. 3A).Specifically, in line 1, there was a difference in sensitivity to oxaliplatin (P < 0.05), but not to SN38 or 5-FU.Line 2 showed differences in sensitivity to SN38 (P < 0.05) only while line 3 showed differences in sensitivity to 5-FU (P < 0.05).Line 4 showed difference in all three drugs (P < 0.05; Supplementary Fig. S2).
We extended these analyses to high-throughput drug screens in three pairs of matched Mycoplasma-negative and -positive lines using a panel of 147 FDA-approved oncology agents.Analysis of these screens pinpointed both similarities and differences in drug sensitivity between pairs of Mycoplasmanegative and -positive lines (Fig. 3B).We plotted the relative drug sensitivity as a differential relative to the Mycoplasma-negative line for each matched organoid pair.These analyses revealed heterogeneous responses to drug depending on Mycoplasma contamination status (Fig. 3C; Supplementary Table S1).
Collectively, these results demonstrate that Mycoplasma contamination can substantially inhibit the growth of organoids and contributes to differential drug response in dose-response assays and high-throughput drug screens.
These analyses highlight the importance of confirming the Mycoplasmanegative status of organoids prior to performing experiments.

Discussion
Mycoplasma are common contaminants in cell cultures (4), and to date Mycoplasma detection and elimination has been extensively studied in cell lines (3,(7)(8)(9)(10)(11). PCR is a common detection method, and can be performed with commercial kits or custom-made primers.Unfortunately, Mycoplasma contamination can be difficult to eradicate as they can be less than 1 μm in size, allowing them to go through filters when most other bacteria would be eliminated walls, they become difficult to eradicate (3).Thus, sterile techniques, filters with small mesh sizes, and autoclaving lab supplies are essential to prevent Mycoplasma contamination in cell culture.However, if Mycoplasma contamination does occur, it must be dealt with quickly and carefully to avoid further spread and contamination.

FIGURE 1
FIGURE 1 Plasmocin cannot reliably clear Mycoplasma from PDO lines.A, Mycoplasma testing of Mycoplasma-positive lines treated with Plasmocin for 2 weeks according to the manufacturer's protocol.The top bands are a negative control for the PCR.The bottom bands are indicative of the presence of Mycoplasma (lanes 1 and 2).The top band may not appear if the original sample contained a high amount of Mycoplasma.All four lines were tested in triplicate.B, Growth rate of PDO4 after Plasmocin treatment over 14 days.Graphs on the left show CTG fluorescence of the PDO taken over the course of 14 days.Images on right compare PDO growth on days 0 and 14.

FIGURE 2
FIGURE 2 Mycoplasma can be successfully cleared from PDO lines by in vivo passaging through immunodeficient mice.A, Mycoplasma test for organoid lines before (+) and after (−) in vivo passaging.Representative positive and negative control PCR products are included within lanes 1 and 2 of the gel on the left.The top band may not appear if the original sample contained a high amount of Mycoplasma.B, Growth comparison of Mycoplasma-positive and -negative lines over 12 days.Images on top compare PDO growth between positive and negative lines on days 0 and 12. Graphs on the bottom show CTG fluorescence of the PDO taken every other day for 12 days.

FIGURE 3
FIGURE 3 Mycoplasma contamination can change organoid drug sensitivity.A, Dose-response curves for four sets of Mycoplasm-positive and -negative lines for three drugs: oxaliplatin, SN38, and 5-FU.*, P < 0.05 (Mann-Whitney).B, High-throughput screens for three sets of Mycoplasm-positive and -negative organoid lines.Percent killing for each drug is indicated by color, with red being the highest percent killing and blue being the lowest.C, Difference in percent killing in high-throughput screen between Mycoplasm-negative and -positive organoid lines.

TABLE 1
Protocol for eliminating Mycoplasma contamination from PDO